Article Effect of Low Addition to As-Forged 304 Stainless for Dental Applications

Ker-Kong Chen 1,2,† , Chih-Yeh Chao 3,†, Jeng-Huey Chen 1,2, Ju-Hui Wu 1,4 , Yen-Hao Chang 5 and Je-Kang Du 1,2,*

1 Department of Dentistry, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan; [email protected] (K.-K.C.); [email protected] (J.-H.C.); [email protected] (J.-H.W.) 2 School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan 3 Department of Mechanical , National Pintung University of Science and , Pingtung 91201, Taiwan; [email protected] 4 Department of Oral Hygiene, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan 5 PhD Program, School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-7-3121101 (ext. 7003) † These authors contribute equally to this work.

Abstract: The aim of this study was to investigate the effect of incorporating low copper (0, 0.5, 1, 1.5, and 2 wt.%) additions into as-forged AISI 304 (304SS). The and mechanical properties of the steel were examined using scanning electron microscopy and a universal testing machine. The antibacterial properties of the Cu-bearing 304SS specimens were investigated using . Each specimen was soaked in artificial saliva to detect the release of copper through inductively coupled plasma atomic emission spectrometry. The addition of copper had no significant effect on the of the as-forged Cu-bearing 304SS, but it slightly increased its maximum tensile strength. The antibacterial rate of the as-cast and as-forged 304SS with   2 wt.% Cu was over 80%, which corresponded to an increase in the release of copper ions. This study demonstrates that low-Cu-content stainless steel can reduce bacteria and can be a suitable material Citation: Chen, K.-K.; Chao, C.-Y.; for the oral environment because of the low release of Cu ions. Chen, J.-H.; Wu, J.-H.; Chang, Y.-H.; Du, J.-K. Effect of Low Copper Keywords: Cu-bearing stainless steel; microstructures; mechanical properties; antibacterial properties Addition to As-Forged 304 Stainless Steel for Dental Applications. Metals 2021, 11, 43. https://dx.doi.org/ 10.3390/met11010043 1. Introduction

Received: 23 November 2020 Orthodontic appliances, orthodontic , and miniscrew implants (MSIs) are com- Accepted: 24 December 2020 mon and are widely used in orthodontic clinics [1,2]. In recent years, MSIs have been Published: 27 December 2020 frequently used as bone anchors for orthodontic treatment. In addition to improving the treatment mode of orthodontics, MSIs can also greatly shorten the treatment time. MSIs are Publisher’s Note: MDPI stays neu- generally made of alloys [3]. Titanium is biocompatible and allows for a high tral with regard to jurisdictional claims bone–implant contact ratio (osseointegration) between dental implants and the host bone in published maps and institutional after healing [4]. From the point of view of the clinician, an ideal MSI requires immedi- affiliations. ate loading after implantation, sufficient mechanical rigidity, and resistance to . After the orthodontic treatment is completed, the MSI must be easy for the clinician to remove without breaking. However, the high degree of osseointegration required for dental

Copyright: © 2020 by the authors. Li- implants is not a requirement for orthodontic MSIs to function as anchorage devices [5]. censee MDPI, Basel, Switzerland. This Unlike titanium alloys, stainless steel (SS) tend to develop primary stability (a article is an open access article distributed mechanical locking interface) between the and bone, which lowers the removal under the terms and conditions of the torque, leading to easier retrieval [6]. Creative Commons Attribution (CC BY) There are four major differences between MSIs and general dental implants. One is license (https://creativecommons.org/ that MSIs placed in the alveolar bone must be partially exposed outside the oral mucosa, licenses/by/4.0/). which increases the risk of infection. Second, MSIs need to be immediately loaded and

Metals 2021, 11, 43. https://dx.doi.org/10.3390/met11010043 https://www.mdpi.com/journal/metals Metals 2021, 11, 43 2 of 8

placed in the alveolar bone. Third, the direction of the force is usually lateral, and the implant needs to be removed after the orthodontic treatment is completed. Fourth, MSIs are usually small in size and diameter. The use of MSIs requires that the existence of multiple in the oral cavity be considered. Colonies of these microorganisms form a biofilm in the oral cavity, which can easily cause bacteria to accumulate and attach to the surfaces of instruments and prostheses, causing inflammation and infection of the soft tissues [7]. Some scholars have pointed out that tissue inflammation, infection, and peri-implant inflammation may increase the failure rate of MSI implantation by 30% in its initial stage [8]. To solve these problems, stainless steel can acquire antibacterial functions through methods including surface treatment [9], amorphous treatment [10], and design [11]. However, as surface spraying can only result in short-term antibacterial effects, alloy design is a promising method for obtaining long-term antibacterial properties [11]. Copper exists in trace amounts in the human body (100–200 mg) [12]. The upper limit of daily copper intake in the human body is 0.7–2 mg/d, depending on the age [12]. A moderate amount of copper can promote the production of collagen, bones, blood vessels, and skin in the human body [11]. However, an excessive intake of copper causes cell damage or death, which harms the human body and causes diseases such as Wilson’s disease and Alzheimer’s disease [13]. Although copper-containing stainless steel has good mechanical and antibacterial properties, it has development potential in the field of biomedical materials that is yet to be exploited. However, the release of copper ions should still be controlled so as to ensure the safety of copper-containing stainless steel for human use. Furthermore, copper has been proven to be an effective antibacterial element [14–17]. Therefore, many studies have been conducted on copper-containing stainless steel. However, because copper is a stable element of austenitic in stainless steel, it has a grain refinement effect. Therefore, the addition of copper can strengthen the mechanical properties of stainless steel [18]. In terms of improving the mechanical properties of stainless steel, [18] and heat treatment [19] are also used to meet the strength requirements in stainless steel for use in different types of medical equipment. Austenitic stainless steel has numerous excellent properties, such as nonmagnetism, high strength, good corrosion resistance [20], and superior workability [12]. It is widely used in industrial applications and is suitable for biomedical applications. The AISI 304 alloy is one of the most popular stainless . Zhang et al. [11] added 3.9 wt% copper to 304 stainless steel. The experimental steel was solution treated and then aged so as to obtain Cu-rich precipitation in the matrix, which afforded the steel a strong antibacterial function against Porphyromonas gingivalis (P. gingivalis). However, the corrosion resistance of copper-containing stainless steel is more controversial. Some studies have reported that copper-added stainless steel is prone to local corrosion phenomena [13], such as [21], stress corrosion [22], and grain boundary corrosion [23]. Tong et al. [24] added 2.5–3.5 wt% copper to 316 stainless steel. Their experimental results showed that 316 copper-containing stainless steel could improve mechanical strength after solution and aging heat treatment. However, the corrosion resistance of 316 stainless steel that contains copper is worse than that of pure 316 stainless steel. Although the precipitation of the Cu-rich phase can improve the antibacterial properties, Hong et al. [14] indicated that the presence of copper precipitates affects the stability of the passive film on the surface of stainless steel and reduces its corrosion resistance. Therefore, the copper content of current, common copper-containing austenitic stainless steel is approximately 3 to 5 wt% [16,24–26]. Even though the biocompatibility of 5 wt% copper with stainless steel is within the allowable range, there are still concerns about excessive dissolution in the oral cavity in the long term. The intake of excessive copper leads to severe damage (), owing to copper-induced oxidative damage in the liver and other tissues [27]. Therefore, in this study, we mainly investigated the antibacterial and mechanical properties of forged 304 Metals 2021, 11, 43 3 of 8

stainless steel by adding a low copper content (0.18, 0.54, 1.05, 1.80, and 2.05 wt%). We also measured the release of copper ions by soaking the materials in artificial saliva.

2. Materials and Methods 2.1. Specimen Preparation In this study, 0.18, 0.54, 1.05, 1.80, and 2.05 wt% copper was added to austenitic 304 stainless steel (Table1), which was provided by the Datian Precision Co., Ltd. (O-TA Company, Shenzhen, China). The alloy was melted in a vacuum melting (Ultimate Materials Technology Co., Ltd., Hsinchu, Taiwan)at 1700 ◦C. The smelted stainless steel was homogenized at 1500 ◦C for 1 h. In addition, forging at 1300 ◦C was performed to produce plates with a thickness of 3 ± 0.1 mm, which was followed by solution treatment and at 880 ◦C for 1 h. The resulting and were all made into standard square tensile test bars of 10 × 10 × 2 mm3 so as to facilitate the subsequent experiments.

Table 1. Chemical composition (wt%) of the experimental Cu-bearing 304 stainless steel.

Specimens Fe Cr Ni Si C Mn S P Cu 304 Bal. 18.14 8.11 0.46 0.02 0.80 0.03 0.04 - 304–0.2Cu Bal. 18.21 8.13 0.43 0.02 0.88 0.03 0.03 0.18 304–0.5Cu Bal. 18.29 8.02 0.53 0.03 0.93 0.03 0.04 0.54 304–1.0Cu Bal. 18.16 8.09 0.55 0.03 0.95 0.02 0.03 1.05 304–1.8Cu Bal. 18.10 8.03 0.50 0.02 0.91 0.03 0.03 1.82 304–2.0Cu Bal. 18.13 8.03 0.51 0.03 0.83 0.03 0.04 2.05

2.2. Microstructure The as-forged specimens were prepared by grinding using a series of 150#- to 1500#- grit SiC . Then, alumina powder (Al2O3) and dioxide (SiO2) were used for . The specimens were corroded in a mixed solution containing 30% nitric (HNO3) + 10% (HCl) + 5% hydrofluoric acid (HF) + 55% for 3 min. Water and alcohol were used to ultrasonically clean the corroded specimens for 10 min. A scanning electron (SEM; JEOL JSM-6380, Tokyo, Japan) was used to observe the microstructure under 15 kV.

2.3. Mechanical Properties The as-forged tensile test specimen in this study was made according to the standard size specifications of the American Society for Testing and Materials. Its neck diameter was 6.25 mm, length was 105 mm, and diameter was 12 mm. Then, a universal testing machine (E45.305, Eden Prairie, MN, USA) was used to perform the tensile test at a rate of 3 mm/min. The was measured using a Vickers Hardness machine (MVK-H11, Akashi, Tokyo, Japan) to randomly measure five specimens under a load of 500 g. Each specimen was measured at three points, and the average was obtained.

2.4. Antibacterial Testing The antibacterial properties of as-cast and as-forged 304 SS and Cu-bearing 304 SS were evaluated according to the JIS Z2801–2000 specifications [28]. The experimental specimens were sterilized using laboratory (MLS-3781L, Tokyo, Japan). Then, 0.4 mL of an Escherichia coli (E. coli, ATCC-25922, Industry Research and Development Institute, Hsinchu, Taiwan) suspension with a of 106 CFU/mL was dripped onto the specimens (with a surface area of 10 × 10 mm2), as well as onto a control group (304 SS). The specimens were washed with -buffered saline after standing in the suspension for 24 h. The bacterial solution was diluted to 106 and 107, and 100 µL was applied to each agar plate. Afterwards, the plates were placed in orbital shaking incubators. The number of remaining colonies on the medium was calculated after 24 h. Metals 2021, 11, 43 Metals 2021, 11, x FOR PEER REVIEW 4 of4 of9 8

ment Institute, Hsinchu, Taiwan) suspension with a density of 106 CFU/mL was dripped The experimentonto was the repeated specimens three (with times, a surface and area the of formula 10 × 10 mm for2), calculatingas well as onto the a control antibacterial group rate was as follows:(304 SS). The specimens were washed with phosphate-buffered saline after standing in the suspension for 24 h. The bacterial solution was diluted to 106 and 107, and 100 μl was applied to each agar plate. Afterwards, the plates were placed in orbital shaking incu- Antibacterial rate (AR) = (N − N )/N × 100% bators. The number of remaining colonies304 on304 the− mediumCu 304was calculated after 24 h. The experiment was repeated three times, and the formula for calculating the antibacterial where N304 andrate N304-Cu was asare follows: the average numbers of bacterial colonies on the 304SS and 304-Cu specimens, respectively. Antibacterial rate (AR)N304 N304-Cu)/N304 100%

2.5. Copper Releasewhere N304 and N304-Cu are the average numbers of bacterial colonies on the 304SS and 304-Cu specimens, respectively. The as-cast and as-forged 304SS and Cu-bearing 304 were immersed in artificial ◦ saliva [29] with2.5. a pH Copper value Ion ofRelease 7.5 at 37 C, per ISO Standard 10993–12:2002, for 1, 2, and 3 days. The concentrationThe as-cast of Cuand as-forged ions released 304SS and was Cu-bea analyzedring 304 usingwere immersed inductively in artificial coupled sa- plasma atomic emissionliva [29] with spectrometry a pH value of (PerkinElmer 7.5 at 37 °C, per Inc.—OptimaISO Standard 10993–12:2002, 2100 DV, Waltham, for 1, 2, and MA, 3 USA) with an accuracydays. The of concentration 0.01 mg/L. of Cu ions released was analyzed using inductively coupled plasma atomic emission spectrometry (PerkinElmer Inc. - Optima 2100 DV, Waltham, 3. Results MA, USA) with an accuracy of 0.01 mg/L.

3.1. Microstructure3. Results Observation Typical SEM3.1. images Microstructure of the Observation alloys subjected to solution treatment are shown in Figure1 . The microstructureTypical of the SEM as-forged images of Cu-bearing the alloys subjected 304SS to was solution a single treatment are shown (γ )in phase. Fig- With the additionure of 1. Cu,The itmicrostructure was found thatof the all as-forged specimens Cu-bearing presented 304SS similarwas a single microstructures, austenite (γ) implying that thephase. Cu additionWith the addition had no of obvious Cu, it was influence found that onall specimens the microstructures. presented similar Moreover, micro- the grain sizes ofstructures, Cu-bearing implying 304SS that with the Cu different addition Cuhad additionsno obvious wereinfluence also on similar, the microstruc- and the tures. Moreover, the grain sizes of Cu-bearing 304SS with different Cu additions were average grain sizes were approximately 25 µm, indicating that a low Cu addition played also similar, and the average grain sizes were approximately 25 μm, indicating that a low no apparent roleCu in addition determining played no the apparent grain size.role in determining the grain size.

FigureFigure 1. SEM 1. imagesSEM of images forged of304 forgedstainless 304 steel stainless with different steel copper with contents: different (a copper) 304–0.2Cu, contents: (b) 304–0.5Cu, (a) 304–0.2Cu, (c) 304–1Cu,(b) 304–0.5Cu,(d) 304–1.8Cu, (andc) 304–1Cu, (e) 304–2Cu. (d ) 304–1.8Cu, and (e) 304–2Cu.

3.2. Mechanical Properties The detailed mechanical properties, including the ultimate tensile strength (UTS), strength (YS), elongation, and hardness under different Cu additions, are shown in

Table2. It is noted that the increase in Cu content had a slight influence on the UTS and YS of Cu-bearing 304SS. The increment in UTS and YS was approximately 30 MPa relative to those of 304SS. However, the addition of Cu had no effect on elongation. The hardness of the Cu-bearing 304SS with different Cu contents showed no obvious variation. Metals 2021, 11, x FOR PEER REVIEW 5 of 9

3.2. Mechanical Properties The detailed mechanical properties, including the ultimate tensile strength (UTS), yield strength (YS), elongation, and hardness under different Cu additions, are shown in Metals 2021, 11, 43 Table 2. It is noted that the increase in Cu content had a slight influence on the UTS and 5 of 8 YS of Cu-bearing 304SS. The increment in UTS and YS was approximately 30 MPa rela- tive to those of 304SS. However, the addition of Cu had no effect on elongation. The hardness of the Cu-bearing 304SS with different Cu contents showed no obvious varia- Table 2. Mechanical properties of the forged 304 stainless steel with different copper contents. tion. Ultimate Tensile Table 2. Mechanical properties of the forged 304 stainless steel with differentYield Strengthcopper contents.Elongation Hardness Specimen Strength (YS, MPa) (%) (Hv) Ultimate Tensile Strength (UTS, MPa) Specimen Yield Strength (YS, MPa) Elongation (%) Hardness (Hv) (UTS, MPa) 304 591.6 355.2 57.1 213.4 304 591.6 304–0.2Cu 355.2 590.2 350.3 57.1 58.4213.4 212.6 304–0.2Cu 590.2 304–0.5Cu 350.3 601.7 368.3 58.4 58.1212.6 213.2 304–0.5Cu 601.7 304–1.0Cu 368.3 627.4 395.2 58.1 55.3213.2 216.4 304–1.0Cu 627.4 304–1.8Cu 395.2 632.8 400.7 55.3 54.9216.4 217.5 304–1.8Cu 632.8 304–2.0Cu 400.7 639.5 411.0 54.9 55.1217.5 220.9 304–2.0Cu 639.5 411.0 55.1 220.9

3.3.3.3. Antibacterial Antibacterial Testing Testing FigureFigure 2 shows2 shows the antibacterial the antibacterial properties properties of the alloy of in the this alloy study in and this E. studycoli co- and E. coli co- culturedcultured for for1 d. 1The d. figure The figure shows showsthat the thatantibacterial the antibacterial rate of cast rate304 and of castforged 304 304 and forged 304 stainlessstainless steel steel increased increased with the with copper the coppercontent. content.This shows This that showshigher copper that higher content copper content resultsresults in better in better antibacterial antibacterial properties, properties, and the antibacterial and the antibacterial properties of properties the two show of the two show thethe same same trend. trend. Among Among them, them,the antibacterial the antibacterial rates of cast rates 304 ofand cast forged 304 304 and stainless forged 304 stainless steelsteel reached reached 82.6% 82.6% and 80.9% and 80.9%for 2 wt.% for Cu 2 wt.% copper Cu content. copper content.

FigureFigure 2. Colonization 2. Colonization of E. coli of E.after coli 24after h coculture 24 h coculture with a change with in aCu change content. in Cu content.

3.4.3.4. Copper Copper Ion Release Ion Release TheThe copper copper ion release ion release results resultsare shown are in shown Figure in3. Regardless Figure3. Regardlessof the forging of or the forging or castingcasting of ofcopper-containing copper-containing 304 stainless 304 stainless steel, the steel, accumulated the accumulated copper ion copper concentra- ion concentration tionincreased increased with with the the copper copper content in in the the physiological physiological environment. environment. The data The also data also revealed revealedthat the that concentration the concentration of released of released Cu Cu ions ions increased increased withwith the immersion immersion time. time. The release Theconcentrations release concentrations of Cu ionsof Cu from ions castfrom andcast forgedand forged 304-Cu 304-Cu specimens specimens werewere 0.13–0.24 and Metals 2021, 11, x FOR PEER REVIEW0.13–0.24 and 0.10–0.19 mg/L over three days, respectively. Furthermore, it is noted 6that of 9 0.10–0.19 mg/L over three days, respectively. Furthermore, it is noted that as-cast 304Cu as-castexhibited 304Cu a exhibited relatively a relatively higher releasehigher release concentration concentration than than the the as-forged. as-forged.

FigureFigure 3. 3.ReleaseRelease concentrations concentrations of copper of copper ions from ions 304C fromu stainless 304Cu steel stainless over 1, steel 2, and over 3 days: 1, 2, (a and) 3 days: (a) as ascast cast and and ((b) as as forged. forged.

4. Discussion Stainless steel is widely used in medical equipment. In the field of dentistry, there are many challenges because of the complex oral environment, especially bacteria in the oral cavity. In addition, medical devices exposed to the oral environment, such as or- thodontic wires, need to withstand tension in order to maintain the orthodontics for ideal positioning of the teeth. Therefore, the combination of antibacterial properties, biocom- patibility, and mechanical properties is very important. Copper is used as an alloying element added to steel. Its main purpose is to improve the mechanical properties, corro- sion resistance, and cold deformation of steel [13,30]. Furthermore, Cu2+ is similar to Ag+, which has a high affinity for the groups contained in some enzymes and amino ac- ids. It can disrupt the bacterial metabolism or generate through reactions by binding to these [31]. Therefore, it can endow stainless steel with an antibacterial ability [32]. In the present study, a low-Cu-bearing 304SS was developed. The microstructures, antibacterial properties, mechanical properties, and Cu ion release concentrations of as-cast and as-forged Cu-bearing 304SS were investigated. A higher Cu content in the passive film lowers the stability of the film because of the reaction of Cu + 2e→Cu2+ and enhances the of the charge transfer [24]. Thus, the increase in Cu content in the passive film significantly decreased the corrosion re- sistance of the Cu-bearing SS. Additionally, copper-containing stainless steel precipitates a Cu-rich phase owing to the supersaturated copper . Ren [12] indicated that 304 stainless steel containing copper precipitates the Cu-rich phase after long-term aging treatment. In addition, different heat treatments can also control the amount of precipi- tates formed and play an important role in the resulting antibacterial properties [33]. However, owing to the formation of a potential difference between the Cu-rich phase and the matrix, the rate is accelerated, and the corrosion resistance of stainless steel is reduced [12,24]. Therefore, in this study, we only used low-copper and solid-solution treatments to maintain corrosion resistance. The addition of approximately 0.2–2.0 wt.% copper has no effect on the microstructure after forging. It was observed from the SEM image that there was no precipitation at the or boundary. Copper is a stable element in the austenitic phase of stainless steel; it can replace and also has a solid solution strengthening effect. Therefore, both UTS and YS increased with the cop- per content and showed similar trends. The antibacterial performance of copper is weaker than that of ; therefore, a relatively high copper content is needed to obtain the required antibacterial performance. However, an upper limit for the safe daily intake of copper exists [34] in the human body; therefore, the addition of copper needs to be carefully considered. In the experimental results for copper ion release in this study, it was found that the release amount of cast 304 stainless steel with low copper (0.2–2.0 wt.%) was higher than that of the forged steel.

Metals 2021, 11, 43 6 of 8

4. Discussion Stainless steel is widely used in medical equipment. In the field of dentistry, there are many challenges because of the complex oral environment, especially bacteria in the oral cavity. In addition, medical devices exposed to the oral environment, such as orthodontic wires, need to withstand tension in order to maintain the orthodontics for ideal position- ing of the teeth. Therefore, the combination of antibacterial properties, biocompatibility, and mechanical properties is very important. Copper is used as an alloying element added to steel. Its main purpose is to improve the mechanical properties, corrosion resistance, and cold deformation of steel [13,30]. Furthermore, Cu2+ is similar to Ag+, which has a high affinity for the thiol groups contained in some enzymes and amino . It can disrupt the bacterial metabolism or generate reactive oxygen species through redox reactions by binding to these thiols [31]. Therefore, it can endow stainless steel with an antibacterial ability [32]. In the present study, a low-Cu-bearing 304SS was developed. The microstruc- tures, antibacterial properties, mechanical properties, and Cu ion release concentrations of as-cast and as-forged Cu-bearing 304SS were investigated. A higher Cu content in the passive film lowers the stability of the oxide film because of the reaction of Cu + 2e→Cu2+ and enhances the flux of the charge transfer [24]. Thus, the increase in Cu content in the passive film significantly decreased the corrosion resistance of the Cu-bearing SS. Additionally, copper-containing stainless steel precipitates a Cu-rich phase owing to the supersaturated copper atoms. Ren [12] indicated that 304 stainless steel containing copper precipitates the Cu-rich phase after long-term aging treatment. In addition, different heat treatments can also control the amount of precipitates formed and play an important role in the resulting antibacterial properties [33]. However, owing to the formation of a potential difference between the Cu-rich phase and the matrix, the galvanic corrosion rate is accelerated, and the corrosion resistance of stainless steel is reduced [12,24]. Therefore, in this study, we only used low-copper and solid-solution treatments to maintain corrosion resistance. The addition of approximately 0.2–2.0 wt.% copper has no effect on the microstructure after forging. It was observed from the SEM image that there was no precipitation at the base or boundary. Copper is a stable element in the austenitic phase of stainless steel; it can replace nickel and also has a solid solution strengthening effect. Therefore, both UTS and YS increased with the copper content and showed similar trends. The antibacterial performance of copper is weaker than that of silver; therefore, a relatively high copper content is needed to obtain the required antibacterial performance. However, an upper limit for the safe daily intake of copper exists [34] in the human body; therefore, the addition of copper needs to be carefully considered. In the experimental results for copper ion release in this study, it was found that the release amount of cast 304 stainless steel with low copper (0.2–2.0 wt.%) was higher than that of the forged steel. A possible reason for this is that after forging, stainless steel can obtain better corrosion resistance than after ; therefore, the copper ions are less likely to be released into the electrolyte. In particular, the highest release of copper ions was only 0.24 mg/L for the alloy used in this study. This is below the upper limit of daily copper intake in the human body. This shows that the alloy has no direct safety concerns. In addition to the antibacterial properties of copper, a low concentration (13.3 µM) of copper is required for proteases in organisms. An appropriate amount of copper can maintain the stable growth of collagen and skin in the human body, and reduce the occurrence of cardiovascular disease [35]. Combining the results of the antibacterial property and Cu ion release tests, it can be deduced that the addition of metallic Cu into the 304-Cu SS could provide bivalent Cu ions, which are key antibacterial agents. Xi et al. [24] explained that when 316L–2.5Cu was solution treated at 1100 ◦C for 30 min and 700 ◦C for 6 h, a higher number and density of spherical Cu-rich precipitates, with a diameter of approximately 10 nm, were homogeneously distributed within the matrix compared with in this study. The alloy had an E. coli antibacterial rate of 94.5%. In addition, Zhang et al. [14] added 3.9 wt% copper to 304 stainless steel followed by a 1040 ◦C solution treatment for 30 min and a 700 ◦C aging treatment for 6 h so as to precipitate the copper-rich phase. P. gingivalis was used for the Metals 2021, 11, 43 7 of 8

antibacterial experiments in their study. The antibacterial properties of copper-containing stainless steel reached 100% after 10–12 h. This shows that the copper-rich phase plays an important role, but that it is necessary to further evaluate the antibacterial and corrosion resistance of the copper-rich phase. Numerous studies have shown that regardless of whether alloys are stainless steel or titanium, the appropriate addition of copper can bring antibacterial properties to the material [28,32,36,37]. However, considering the actual application conditions, the oral environment is complex, and the material is soaked in saliva for a long time. Thus, antibacterial properties that are too great (large amounts of Cu ions released) may not be suitable for oral biomedical materials. In addition, a large copper addition and long- term heat treatment will increase the cost of production. Therefore, it may be possible to modify the alloy design at the application level in order to meet the needs of clinical use in the development of medical equipment. This study confirms that cast or forged stainless steel with a 2 wt.% Cu content can reach an antibacterial rate of more than 80%, indicating that a low copper content can still have distinct advantages and can maintain a balanced relationship between antibacterial properties and corrosion resistance.

5. Conclusions The addition of Cu in the present study had no influence on the microstructure of the as-forged 304SS. The YS and UTS of the forged 304-Cu SS was increased under different Cu contents, with corresponding solution strengthening. The antibacterial rate of the as-forged 304SS with 2 wt.% Cu was over 80%, which corresponded to an increased release of copper ions. The amount of copper ions released showed no toxicity to humans in the oral environment. The current study indicates that antibacterial Cu-bearing 304 SS is a potential material for the oral environment.

Author Contributions: Conceptualization, K.-K.C., J.-H.C., and C.-Y.C.; methodology, J.-K.D. and C.-Y.C.; software, J.-H.W. and Y.-H.C.; validation, J.-K.D. and J.-H.C.; formal analysis, C.-Y.C. and K.-K.C.; investigation, J.-K.D. and Y.-H.C.; resources, J.-H.C. and J.-H.W.; data curation, J.-H.W.; writing—original preparation, J.-K.D., Y.-H.C., and C.-Y.C.; writing—review and editing, J.-K.D., C.-Y.C., and Y.-H.C.; visualization, J.-H.C.; supervision, K.-K.C.; project administration, K.-K.C.; funding acquisition, K.-K.C. and J.-K.D. All authors have read and agreed to the published version of the manuscript. Funding: This study was financially supported by funding from the Ministry of Science and Tech- nology (MOST 106–2314-B-037–013 and MOST 107–2314-B-037–46), and the Kaohsiung Medical University Hospital (KMUH104-4M47, KMUH 106–6R73 and KMUH 108–8M62). Conflicts of Interest: The authors declare no conflict of interest.

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